Gene expressed in prostate cancer and methods of use

ABSTRACT

A new polypeptide is disclosed that is specifically detected in the cells of the prostate, termed Novel Gene Expressed in Prostate (NGEP). Polynucleotides encoding NGEP are also disclosed, as are vectors including these polynucleotides. Host cells transformed with these polynucleotides are also disclosed. Antibodies are disclosed that specifically bind NGEP. Methods are disclosed for using an NGEP polypeptide, an antibody that specifically binds NGEP, or a polynucleotide encoding NGEP. Assays are disclosed for the detection prostate cancer. Pharmaceutical compositions including an NGEP polypeptide, an antibody that specifically binds NGEP, or a polynucleotide encoding NGEP are also disclosed. These pharmaceutical compositions are of use in the treatment of prostate cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/495,663, filed on May 12, 2004, which is the § 371 U.S. NationalStage of PCT Application No. PCT/US2002/036648, filed Nov. 13, 2002,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Application No. 60/336,308, filedNov. 14, 2001. The prior applications are incorporated by referenceherein in their entirety.

FIELD OF THE INVENTION

This disclosure relates the prostate, specifically to polypeptidesexpressed specifically in the prostate. The disclosure further relatesto detection and treatment of prostate cancer.

BACKGROUND OF THE INVENTION

Cancer of the prostate is the most commonly diagnosed cancer in men andis the second most common cause of cancer death (Carter and Coffey,Prostate 16:39-48, 1990; Armbruster et al., Clinical Chemistry 39:181,1993). If detected at an early stage, prostate cancer is potentiallycurable. However, a majority of cases are diagnosed at later stages whenmetastasis of the primary tumor has already occurred (Wang et al., Meth.Cancer Res. 19:179, 1982). Even early diagnosis is problematic becausenot all individuals who test positive in these screens develop cancer.

Prostate specific antigen (PSA) is a 240 amino acid member of theglandular kallikrein gene family. (Wang et al., 1982, supra; Wang etal., Invest. Urology, 17:159, 1979; Bilhartz et al., Urology, 38:95,1991). PSA is a serine protease, produced by normal prostatic tissue,and secreted exclusively by the epithelial cells lining prostatic aciniand ducts (Wang et al., 1982, supra; Wang et al., 1979, supra; Lilja etal., World J. Urol., 11:188-191, 1993). Prostate specific antigen can bedetected at low levels in the sera of healthy males without clinicalevidence of prostate cancer. However, during neoplastic states,circulating levels of this antigen increase dramatically, correlatingwith the clinical stage of the disease (Schellhammer et al., UrologicClinics of North America 20:597, 1993; Huang et al., Prostate 23:201,1993). Prostate specific antigen is now the most widely used marker forprostate cancer. However, there clearly is a need to identify additionalantigens to aid in the diagnosis of prostate cancer, and for use astherapeutic agents.

Present treatment for prostate cancer includes radical prostatectomy,radiation therapy, or hormonal therapy. With surgical intervention,complete eradication of the tumor is not always achieved and theobserved re-occurrence of the cancer (12-68%) is dependent upon theinitial clinical tumor stage (Zietman et al., Cancer 71:959, 1993).Thus, alternative methods of treatment including prophylaxis orprevention are desirable.

Immunotherapy is a potent new weapon against cancer. Immunotherapyinvolves evoking an immune response against cancer cells based on theirproduction of target antigens. Immunotherapy based on cell-mediatedimmune responses involves generating a cell-mediated response to cellsthat produce particular antigenic determinants, while immunotherapybased on humoral immune responses involves generating specificantibodies to cells that produce particular antigenic determinants.

Cancer cells produce various proteins that can become the target ofimmunotherapy; antigenic determinants normally present on a specificcell type can also be immunogenic. For example, Rosenberg et al. haveshown that tumor infiltrating lymphocytes target and recognize antigenicdeterminants of the protein MART-1, produced by both normal melanocytesand malignant melanoma cells. Furthermore, active or passiveimmunotherapy directed against MART-1 or peptides of it that bind to MHCClass I molecules (epitopes of HLA A2, in particular) results in thedestruction of melanoma cells as well as normal cells that produceMART-1 (Kawakami et al., J. Immunol. 21:237, 1998). The tissuespecificity of PSA has made it a potential target antigen for activespecific immunotherapy (Armbruster et al., Clin. Chemistry 39:181, 1993;Brawer et al., Cancer Journal Clinic 39:361, 1989), especially inpatients who have undergone a radical prostatectomy in which the onlyPSA expressing tissue in the body should be in metastatic deposits.

Recent studies using in-vitro immunization have shown the generation ofCD4 and CD8 cells specific for PSA (Peace et al., Cancer Vaccines:Structural Basis for Vaccine Development (Abstract), 1994; Correale etal., 9th International Congress of Immunology (Abstract), 1995), andmethods for inducing an immune response against PSA include the use ofviral vectors incorporating DNA encoding PSA (e.g. see U.S. Pat. No.6,165,460; Hodge et al., Cancer 63:231, 1995). Discovery of additionalantigens expressed by the prostate gland can similarly be used to designimmunotherapy methods for prostate cancer.

SUMMARY OF THE INVENTION

A new polypeptide is disclosed herein that is specifically detected inthe cells of the prostate. This polypeptide is termed Novel GeneExpressed in Prostate (NGEP). Polynucleotides encoding NGEP are alsodisclosed herein, as are vectors including polynucleotides encodingNGEP, and host cells transformed with these polynucleotides. Antibodiesthat specifically bind NGEP are also disclosed.

Methods for using an NGEP polypeptide, an antibody that specificallybinds NGEP, or a polynucleotide encoding NGEP are also disclosed. Aspecific, non-limiting example of a method of use is an assay to detectprostate cancer. Also disclosed are pharmaceutical compositionsincluding an NGEP polypeptide, an antibody that specifically binds NGEP,or a polynucleotide encoding NGEP. In one embodiment, the pharmaceuticalcomposition is used to treat prostate cancer.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the CW-1 cluster, which was identifiedby computer analysis of the human EST database as a prostate specificcluster. There are 5 ESTs in this cluster of which 3 ESTs are fromnormal prostate and two ESTs are from prostate cancer (FIG. 1). All ESTsare localized at chromosome 2q37.3 on human genome. Pr4.1: Prostatecancer; Pr22: normal prostate; Pr28 normal prostate; Pr23 Prostatecancer.

FIG. 2 is a set of digital images showing tissue specific expression ofNGEP mRNA. FIG. 2A is a digital image of a RNA hybridization of amultiple tissue dot-blot containing mRNA from 61 normal human cell typesor tissues using NGEP cDNA as probe. NGEP expression is observed only inprostate (E8) and no detectable expression in brain (A1), heart (A4),lung (A8), kidney (A7), and pancreas (B9). FIG. 2B is a digital image ofPCR on cDNAs from a panel of cDNAs isolated from several normal tissuesincluding brain, heart, liver, lung, pancreas, and colon. The expectedsize of the NGEP PCR product is 176 bp. Lanes are: 1, no DNA; 2, DU145;3, normal prostate; 4, prostate cancer; 5, prostate cancer; 6, brain; 7,heart; 8, kidney; 9, liver; 10, lung; 11, skeletal muscle; 12, normalprostate; 13, colon; 14, peripheral blood leukocyte; 15, smallintestine; 16, ovary; 17, spleen; 18, thymus; 19, testis; 20, pancreas;and 21, placenta. FIG. 2C is a digital image of a Northern blot analysisof NGEP in different normal tissues. The matured transcript (shown by anarrow) is about 1.0 kb in size and is expressed only in prostate. Thesignal at the high molecular weight region is probably from theunspliced NGEP transcript. FIG. 2D is a digital image of aRT-PCRanalysis of RNAs from prostate cancer cell line LnCAP and PC3. Lanes: M,molecular weight standard; 1, negative control; 2, PC3, 3, LnCAP; and 4,pNGEP plasmid positive control.

FIG. 3 shows the amino acid sequence of NGEP (SEQ ID NO:1). NGEP is 179amino acids in length, and has a predicted molecular weight of 19.6 kDa.Analysis of the amino acid sequence of NGEP protein was performed usingthe PROSITE program. Three possible casein kinase II phosphorylationsites are shown in rectangular boxes. One potential protein kinase Cphosphorylation site is marked with one line whereas, the predictedcamp-dependent protein kinase site is marked with two lines.

FIG. 4 is a set of digital images of in situ localization of NGEP mRNAin prostate tissue. The image labeled “H/E” is a digital image of abenign prostate section stained with H/E to show the morphology. Theepithelial cells are stained blue while the connective tissue is pink.In the digital image labeled CD22, B lymphocyte specific gene CD22 usedas a probe for negative control for in situ hybridization. Note theabsence of signal in the epithelial cells. In the digital image labeled“U6,” a small nuclear RNA was used as a probe for positive control of insitu hybridization. The epithelial cells show a strong signal. In thedigital image labeled “NGEP” a NGEP sequence was used as probe forlocalizing the region of its expression in the prostate tissue section.NGEP also shows strong signal in the epithelial cells.

FIG. 5 is a set of digital images of the analysis and localization ofprotein product encoded by NGEP. FIG. 5A is a digital image of in vitrotranslation of the NGEP cDNA. Expected 20 kDa protein product isdetected in lane with NGEP cDNA. Lane with vector DNA alone showed nodetectable bands. FIG. 5B is a digital image of a Western blot analysisof NGEP-transfected cell extract with anti-Myc-tag antibody. Twospecific bands (20 and 24 kDa) are detected in cell extracts transfectedwith plasmid pNGEP-Myc. Cell extract from pPATE-Myc transfected cells isused as positive control and detects an expected size band of 16 kDa.FIGS. 5C and 5D are digital images of immunocytochemical analysis ofNGEP localization in transfected cells. Immunocytochemistry usinganti-Myc antibody was done under confocal microscopy in LnCAP cellsco-transfected with pEGFP and pNGEP-Myc. The signals representing NGEPexpression were localized in both cytoplasm and nucleus (FIG. 5C). In asmall fraction of positive cells (<5%), positive signals were foundexclusively in cytoplasm (FIG. 5D).

FIG. 6 is the nucleotide sequence (SEQ ID NO: 2) of the full-length (917nucleotide) NGEP cDNA. The ORF is indicated in bold, and the poly A siteis underlined.

SEQUENCE LISTING

The nucleic and amino acid sequences disclosed herein are shown usingstandard letter abbreviations for nucleotide bases, and three lettercode for amino acids, as defined in 37 C.F.R. 1.822. Only one strand ofeach nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. In theaccompanying sequence listing:

SEQ ID NO:1 is the polypeptide sequence of an NGEP polypeptide.

SEQ ID NO:2 is the polynucleotide sequence of a polynucleotide encodingNGEP.

SEQ ID NO:3 is the sequence of the primer CW74.

SEQ ID NO:4 is the sequence of the primer CW75.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

A novel gene product expressed in cells of the normal prostate andprostate cancer, termed Novel Gene Expressed in Prostate (NGEP), isdisclosed herein.

After defining some of the terms used herein, the discussion below setsforth the discovery of the nature of the NGEP protein, nucleic acidsequences encoding NGEP, and the expression of this protein. As NGEP isexpressed in prostate cancer, it is of use in detecting prostate cancercells. Diagnostic kits for NGEP are thus disclosed.

Antibodies that specifically bind NGEP are also disclosed herein. Theseantibodies are of use in detection assays, as well as in the productionof immunoconjugates, such as immunotoxins, which can be used to targetprostate cancer.

Nucleic acids encoding NGEP, or an NGEP polypeptide, can be used toproduce an immune response against prostate cancer cells. Thus,pharmaceutical compositions including NGEP, or a nucleic acid encodingNGEP are also disclosed.

Terms

The following terms are provided to better define the present disclosureand to guide those of ordinary skill in the art in the practice of themethods described herein. Definitions of common terms can be found inRieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991; and Lewin, Genes V, OxfordUniversity Press: New York, 1994. The standard one- and three letternomenclature for amino acid residues is used.

Additional explanations of terms commonly used in molecular genetics canbe found in Benjamin Lewin, Genes V published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T-cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. Epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue.Specific, non-limiting examples of a tissue specific antigen are aprostate specific antigen. A disease-specific antigen is expressedcoincidentally with a disease process. Specific non-limiting examples ofa disease-specific antigen are an antigen whose expression correlateswith, or is predictive of, tumor formation, such as, prostate cancer. Adisease specific antigen may be an antigen recognized by T cells or Bcells.

Amplification: Of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a specimen. An example of amplification is thepolymerase chain reaction, in which a biological sample collected from asubject is contacted with a pair of oligonucleotide primers, underconditions that allow for the hybridization of the primers to a nucleicacid template in the sample. The primers are extended under suitableconditions, dissociated from the template, and then re-annealed,extended, and dissociated to amplify the number of copies of the nucleicacid. The product of amplification may be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the VL, VH, CL and CH1domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii)an Fv fragment consisting of the VL and VH domains of a single arm of anantibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989)which consists of a VH domain; (v) an isolated complementaritydetermining region (CDR); and (vi) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. No.4,745,055; U.S. Pat. No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694;EP 125, 023; Faoulkner et al., Nature 298:286, 1982; Morrison, J.Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease an activityor antigenicity of NGEP. Specific, non-limiting examples of aconservative substitution include the following examples:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; LeuThe term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide. Non-conservative substitutions are thosethat reduce an activity or antigenicity.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Degenerate variant: A polynucleotide encoding an NGEP polypeptide thatincludes a sequence that is degenerate as a result of the genetic code.There are 20 natural amino acids, most of which are specified by morethan one codon. Therefore, all degenerate nucleotide sequences areincluded in the invention as long as the amino acid sequence of the NGEPpolypeptide encoded by the nucleotide sequence is unchanged.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters, are included (see e.g., Bitter etal., Methods in Enzymology 153:516-544, 1987). For example, when cloningin bacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In one embodiment, an immune response is a T cell response,such as a CD4+ response or a CD8+ response. In another embodiment, theresponse is a B cell response, and results in the production of specificantibodies.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody. The effector molecule can be an immunotoxin. Specific,non-limiting examples of toxins include, but are not limited to, abrin,ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40),diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, orother toxic agents that directly or indirectly inhibit cell growth orkill cells. For example, PE and DT are highly toxic compounds thattypically bring about death through liver toxicity. PE and DT, however,can be modified into a form for use as an immunotoxin by removing thenative targeting component of the toxin (e.g., domain Ia of PE and the Bchain of DT) and replacing it with a different targeting moiety, such asan antibody. A “chimeric molecule” is a targeting moiety, such as aligand or an antibody, conjugated (coupled) to an effector molecule. Theterm “conjugated” or “linked” refers to making two polypeptides into onecontiguous polypeptide molecule. In one embodiment, an antibody isjoined to an effector molecule (EM). In another embodiment, an antibodyjoined to an effector molecule is further joined to a lipid or othermolecule to a protein or peptide to increase its half-life in the body.The linkage can be either by chemical or recombinant means. In oneembodiment, the linkage is chemical, wherein a reaction between theantibody moiety and the effector molecule has produced a covalent bondformed between the two molecules to form one molecule. A peptide linker(short peptide sequence) can optionally be included between the antibodyand the effector molecule.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response(e.g. antibody production) against the antigen from which theimmunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunogenic composition: A composition comprising an NGEP polypeptidethat induces a measurable CTL response against cells expressing NGEPpolypeptide, or induces a measurable B cell response (e.g. production ofantibodies that specifically bind NGEP) against an NGEP polypeptide. Itfurther refers to isolated nucleic acids encoding an NGEP polypeptidethat can be used to express the NGEP polypeptide (and thus be used toelicit an immune response against this polypeptide). For in vitro use,the immunogenic composition may consist of the isolated protein orpeptide. For in vivo use, the immunogenic composition will typicallycomprise the protein or peptide in pharmaceutically acceptable carriers,and/or other agents. Any particular peptide, NGEP polypeptide, ornucleic acid encoding the polypeptide, can be readily tested for itsability to induce a CTL or B cell response by art-recognized assays.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B-cellsand T-cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Open reading frame (ORF): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Peptide: A chain of amino acids of between 3 and 30 amino acids inlength. In one embodiment, a peptide is from about 10 to about 25 aminoacids in length. In yet another embodiment, a peptide is from about 11to about 20 amino acids in length. In yet another embodiment, a peptideis about 12 amino acids in length.

Peptide Modifications: NGEP polypeptides include synthetic embodimentsof peptides described herein. In addition, analogues (non-peptideorganic molecules), derivatives (chemically functionalized peptidemolecules obtained starting with the disclosed peptide sequences) andvariants (homologs) of these proteins can be utilized in the methodsdescribed herein. Each polypeptide of the invention is comprised of asequence of amino acids, which may be either L- and/or D-amino acids,naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of an NGEP polypeptide havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologicallyneutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is NGEPpolypeptide.

Probes and primers: A probe comprises an isolated nucleic acid attachedto a detectable label or reporter molecule. Primers are short nucleicacids, preferably DNA oligonucleotides 15 nucleotides or more in length.Primers may be annealed to a complementary target DNA strand by nucleicacid hybridization to form a hybrid between the primer and the targetDNA strand, and then extended along the target DNA strand by a DNApolymerase enzyme. Primer pairs can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art. One of skillin the art will appreciate that the specificity of a particular probe orprimer increases with its length. Thus, for example, a primer comprising20 consecutive nucleotides will anneal to a target with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers may be selectedthat comprise 20, 25, 30, 35, 40, 50 or more consecutive nucleotides.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription. Both constitutive and inducible promoters are included(see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the retrovirus long terminal repeat; theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used.Promoters produced by recombinant DNA or synthetic techniques may alsobe used. A polynucleotide can be inserted into an expression vector thatcontains a promoter sequence which facilitates the efficienttranscription of the inserted genetic sequence of the host. Theexpression vector typically contains an origin of replication, apromoter, as well as specific nucleic acid sequences that allowphenotypic selection of the transformed cells

Protein Purification The NGEP polypeptides disclosed herein can bepurified by any of the means known in the art. See, e.g., Guide toProtein Purification, ed. Deutscher, Meth. Enzymol. 185, Academic Press,San Diego, 1990; and Scopes, Protein Purification: Principles andPractice, Springer Verlag, New York, 1982. Substantial purificationdenotes purification from other proteins or cellular components. Asubstantially purified protein is at least 60%, 70%, 80%, 90%, 95% or98% pure. Thus, in one specific, non-limiting example, a substantiallypurified protein is 90% free of other proteins or cellular components.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50% of the total peptide or protein contentof the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (e.g., Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, e.g., high stringency conditions, or each of theconditions can be used, e.g., for 10-15 minutes each, in the orderlisted above, repeating any or all of the steps listed. However, asmentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologues or variants of an NGEP polypeptide will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp,CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881,1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988.Altschul et al., Nature Genet., 6:119, 1994 presents a detailedconsideration of sequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologues and variants of an NGEP polypeptide are typicallycharacterized by possession of at least 75%, for example at least 80%,sequence identity counted over the full length alignment with the aminoacid sequence of NGEP using the NCBI Blast 2.0, gapped blastp set todefault parameters. For comparisons of amino acid sequences of greaterthan about 30 amino acids, the Blast 2 sequences function is employedusing the default BLOSUM62 matrix set to default parameters, (gapexistence cost of 11, and a per residue gap cost of 1). When aligningshort peptides (fewer than around 30 amino acids), the alignment shouldbe performed using the Blast 2 sequences function, employing the PAM30matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologues andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologues could be obtained that fall outside ofthe ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus an NGEP specific binding agent is an agent thatbinds substantially to an NGEP polypeptide. In one embodiment, thespecific binding agent is a monoclonal or polyclonal antibody thatspecifically binds NGEP.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human veterinary subjects, including human and non-humanmammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell isa suppressor T cell.

Therapeutically active polypeptide: An agent, such as an NGEPpolypeptide that causes induction of an immune response, as measured byclinical response (for example increase in a population of immune cells,production of antibody that specifically binds NGEP, or measurablereduction of tumor burden). Therapeutically active molecules can also bemade from nucleic acids. Examples of a nucleic acid basedtherapeutically active molecule is a nucleic acid sequence that encodesan NGEP polypeptide, wherein the nucleic acid sequence is operablylinked to a control element such as a promoter. Therapeutically activeagents can also include organic or other chemical compounds that mimicthe effects of NGEP.

The terms “therapeutically effective fragment of NGEP” or“therapeutically effective variant of NGEP” includes any fragment ofNGEP, or variant of NGEP, that retains a function of NGEP, or retains anantigenic epitope of NGEP.

In one embodiment, a therapeutically effective amount of a fragment ofNGEP is an amount used to generate an immune response, or to treatprostate cancer in a subject. Specific, non-limiting examples are theN-terminal half of NGEP or the C-terminal half of NGEP. Treatment refersto a therapeutic intervention that ameliorates a sign or symptom ofprostate cancer, or a reduction in tumor burden.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. “Comprise” means“include.” It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalents tothose described herein can be used in the practice or testing of thepresent disclosure, suitable methods and materials are described below.In case of conflict, the present specification, including explanationsof terms, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

NGEP Polynucleotides and Polypeptides

Substantially purified Novel Gene Expressed in Prostate (NGEP)polypeptides are disclosed herein. In one embodiment, an NGEPpolypeptide has a sequence 75%, 85%, 90%, 95%, or 99% homologous to theamino acid sequence set forth in SEQ ID NO:1 (see FIG. 5). In anotherembodiment, an NGEP polypeptide has a sequence as set forth a SEQ IDNO:1 or is a conservative variant of SEQ ID NO:1. In a furtherembodiment, an NGEP polypeptide has a sequence as set forth as SEQ IDNO:1. Fragments and variants of an NGEP polypeptide can readily beprepared by one of skill in the art using molecular techniques. In oneembodiment, a fragment of an NGEP polypeptide includes at least 8, 10,15, or 20 consecutive amino acids of an NGEP polypeptide. In anotherembodiment, a fragment of an NGEP polypeptide includes a specificantigenic epitope found on full-length NGEP.

One skilled in the art, given the disclosure herein, can purify NGEPpolypeptide using standard techniques for protein purification. Thesubstantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. The purity of the NGEP polypeptide canalso be determined by amino-terminal amino acid sequence analysis.

Minor modifications of the NGEP polypeptide primary amino acid sequencesmay result in peptides which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein.

Thus, specific, non-limiting examples of an NGEP polypeptide is aconservative variant of NGEP. A table of conservative substitutions isprovided herein. Substitutions of the amino acids sequence shown in SEQID NO:1 can be made based on this table. Thus, one non-limiting exampleof a conservative variant is substitution of amino acid one (Met) of SEQID NO: 1 with an arginine residue. Similarly, another non-limitingexample is substitution of amino acid 2 (Arg) of SEG ID NO: 1 with alysine residue. Using the sequence provided as SEQ ID NO: 1, and thedescription of conservative amino acid substitutions provided, one ofskill in the art can readily ascertain sequences of conservativevariants. In several embodiments, a conservative variant includes atmost one, at most two, at most five, at most ten, or at most fifteenconservative substitutions of the sequence shown in SEQ ID NO:1.Generally, a conservative variant will bind to antibodies thatimmunoreact with a polypeptide having a sequence set forth as SEQ IDNO:1.

One of skill in the art can readily produce fusion proteins including anNGEP polypeptide and a second polypeptide of interest. Optionally, alinker can included between the NGEP polypeptide and the secondpolypeptide of interest. Fusion proteins include, but are not limitedto, a polypeptide including an NGEP polypeptide and a marker protein. Inone embodiment, the marker protein can be used to identify or purify anNGEP polypeptide. Exemplary fusion proteins included, but are notlimited to green fluorescent protein, six histidine residues, or myc andan NGEP polypeptide.

Polynucleotides encoding an NGEP polypeptide are also provided, and aretermed NGEP polynucleotides. These polynucleotides include DNA, cDNA andRNA sequences which encode NGEP. It is understood that allpolynucleotides encoding an NGEP polypeptide are also included herein,as long as they encode a polypeptide with the recognized activity, suchas the binding to an antibody that recognizes an NGEP polypeptide. Thepolynucleotides of the invention include sequences that are degenerateas a result of the genetic code. There are 20 natural amino acids, mostof which are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included in the invention as long as the aminoacid sequence of the NGEP polypeptide encoded by the nucleotide sequenceis functionally unchanged. One specific, non-limiting example of apolynucleotide encoding NGEP is SEQ ID NO:2. Another specificnon-limiting example of a polynucleotide encoding NGEP is apolynucleotide having at least 75%, 85%, 90%, 95%, or 99% homologous toSEQ ID NO:2 that encodes a polypeptide having an antigenic epitope orfunction of NGEP. Yet another specific non-limiting example of apolynucleotide encoding NGEP is a polynucleotide that encodes apolypeptide that is specifically bound by an antibody that specificallybinds SEQ ID NO:1.

The NGEP polynucleotides include a recombinant DNA which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA) independent of other sequences. Thenucleotides of the invention can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA. Also included in the inventionare fragments of the above-described nucleic acid sequences that are andare at least 15 bases in length, which is sufficient to permit thefragment to selectively hybridize to DNA that encodes the disclosed NGEPpolypeptide (e.g. a polynucleotide that encodes SEQ ID NO:1) underphysiological conditions. The term “selectively hybridize” refers tohybridization under moderately or highly stringent conditions, whichexcludes non-related nucleotide sequences. The NGEP polynucleotidesequence disclosed herein include, but are not limited to, SEQ ID NO:2,degenerate variants of SEQ ID NO:2, and sequences that encodeconservative variations of NGEP polypeptide.

DNA sequences encoding NGEP polypeptide can be expressed in vitro by DNAtransfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

NGEP polynucleotide sequences can be operatively linked to expressioncontrol sequences. An expression control sequence operatively linked toa coding sequence is ligated such that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding NGEP may be inserted into anexpression vector including, but not limited to a plasmid, virus orother vehicle that can be manipulated to allow insertion orincorporation of sequences and can be expressed in either prokaryotes oreukaryotes. Hosts can include microbial, yeast, insect and mammalianorganisms. Methods of expressing DNA sequences having eukaryotic orviral sequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with NGEP polynucleotide sequences, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

Isolation and purification of recombinantly expressed polypeptide may becarried out by conventional means including preparative chromatographyand immunological separations.

Antibodies

An NGEP polypeptide or a fragment or conservative variant thereof can beused to produce antibodies which are immunoreactive or bind to anepitope of NGEP. Polyclonal antibodies, antibodies which consistessentially of pooled monoclonal antibodies with different epitopicspecificities, as well as distinct monoclonal antibody preparations areincluded.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., “Production of PolyclonalAntisera, in: Immunochemical Protocols pages 1-5, Manson, ed., HumanaPress 1992; Coligan et al., “Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, 1992.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature 256:495, 1975; Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., in: Antibodies: a LaboratoryManual, page 726, Cold Spring Harbor Pub., 1988. Briefly, monoclonalantibodies can be obtained by injecting mice with a compositioncomprising an antigen, verifying the presence of antibody production byremoving a serum sample, removing the spleen to obtain B lymphocytes,fusing the B lymphocytes with myeloma cells to produce hybridomas,cloning the hybridomas, selecting positive clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Monoclonal antibodies can be isolated and purifiedfrom hybridoma cultures by a variety of well-established techniques.Such isolation techniques include affinity chromatography with Protein-ASepharose, size-exclusion chromatography, and ion-exchangechromatography. See, e.g., Coligan et al., sections 2.7.1-2.7.12 andsections 2.9.1-2.9.3; Barnes et al., Purification of Immunoglobulin G(IgG), in: Methods in Molecular Biology, Vol. 10, pages 79-104, HumanaPress, 1992.

Methods of in vitro and in vivo multiplication of monoclonal antibodiesare well known to those skilled in the art. Multiplication in vitro maybe carried out in suitable culture media such as Dulbecco's ModifiedEagle Medium or RPMI 1640 medium, optionally supplemented by a mammalianserum such as fetal calf serum or trace elements and growth-sustainingsupplements such as normal mouse peritoneal exudate cells, spleen cells,thymocytes or bone marrow macrophages. Production in vitro providesrelatively pure antibody preparations and allows scale-up to yield largeamounts of the desired antibodies. Large-scale hybridoma cultivation canbe carried out by homogenous suspension culture in an airlift reactor,in a continuous stirrer reactor, or in immobilized or entrapped cellculture. Multiplication in vivo may be carried out by injecting cellclones into mammals histocompatible with the parent cells, e.g.,syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. After oneto three weeks, the desired monoclonal antibody is recovered from thebody fluid of the animal.

Antibodies can also be derived from subhuman primate antibody. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in WO 91/11465, 1991, and Losman et al., Int. J.Cancer 46:310, 1990.

Alternatively, an antibody that specifically binds an NGEP polypeptidecan be derived from a humanized monoclonal antibody. Humanizedmonoclonal antibodies are produced by transferring mouse complementaritydetermining regions from heavy and light variable chains of the mouseimmunoglobulin into a human variable domain, and then substituting humanresidues in the framework regions of the murine counterparts. The use ofantibody components derived from humanized monoclonal antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions. General techniques for cloning murine immunoglobulinvariable domains are described, for example, by Orlandi et al., Proc.Nat'l Acad. Sci. USA 86:3833, 1989. Techniques for producing humanizedmonoclonal antibodies are described, for example, by Jones et al.,Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Verhoeyenet al., Science 239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci.USA 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singeret al., J. Immunol. 150:2844, 1993.

Antibodies can be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, for example, Barbas et al.,in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 119,1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE CloningSystems (La Jolla, Calif.).

In addition, antibodies can be derived from a human monoclonal antibody.Such antibodies are obtained from transgenic mice that have been“engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994;and Taylor et al., Int. Immunol. 6:579, 1994.

Antibodies include intact molecules as well as fragments thereof, suchas Fab, F(ab′)₂, and Fv which are capable of binding the epitopicdeterminant. These antibody fragments retain some ability to selectivelybind with their antigen or receptor and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing thevariable region of the light chain and the variable region of the heavychain expressed as two chains; and

(5) Single chain antibody (SCA), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratoy Manual, Cold SpringHarbor Laboratory, New York, 1988). An epitope is any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody by expression in E. coli of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)₂. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly (see U.S. Pat. No. 4,036,945 and No. 4,331,647, andreferences contained therein; Nisonhoff et al., Arch. Biochem. Biophys.89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methodsin Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan etal. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent (Inbar et al., Proc. Nat'lAcad. Sci. USA 69:2659, 1972). Alternatively, the variable chains can belinked by an intermolecular disulfide bond or cross-linked by chemicalssuch as glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fvfragments comprise V_(H) and V_(L) chains connected by a peptide linker.These single-chain antigen binding proteins (sFv) are prepared byconstructing a structural gene comprising DNA sequences encoding theV_(H) and V_(L) domains connected by an oligonucleotide. The structuralgene is inserted into an expression vector, which is subsequentlyintroduced into a host cell such as E. coli. The recombinant host cellssynthesize a single polypeptide chain with a linker peptide bridging thetwo V domains. Methods for producing sFvs are known in the art (seeWhitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Pat. No.4,946,778; Pack et al., Bio/Technology 11:1271, 1993; and Sandhu,supra).

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (Larrick et al., Methods: aCompanion to Methods in Enzymology, Vol. 2, page 106, 1991).

Antibodies can be prepared using an intact polypeptide or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or a peptide used to immunize an animal can be derived fromsubstantially purified polypeptide produced in host cells, in vitrotranslated cDNA, or chemical synthesis which can be conjugated to acarrier protein, if desired. Such commonly used carriers which arechemically coupled to the peptide include keyhole limpet hemocyanin(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.The coupled peptide is then used to immunize the animal (e.g., a mouse,a rat, or a rabbit).

Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coliganet al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region that is the“image” of the epitope bound by the first mono-clonal antibody.

Binding affinity for a target antigen is typically measured ordetermined by standard antibody-antigen assays, such as competitiveassays, saturation assays, or immunoassays such as ELISA or RIA. Suchassays can be used to determine the dissociation constant of theantibody. The phrase “dissociation constant” refers to the affinity ofan antibody for an antigen. Specificity of binding between an antibodyand an antigen exists if the dissociation constant (K_(D)=1/K, where Kis the affinity constant) of the antibody is, for example <1 μM, <100nM, or <0.1 nM. Antibody molecules will typically have a K_(D) in thelower ranges. K_(D)=[Ab-Ag]/[Ab][Ag] where [Ab] is the concentration atequilibrium of the antibody, [Ag] is the concentration at equilibrium ofthe antigen and [Ab-Ag] is the concentration at equilibrium of theantibody-antigen complex. Typically, the binding interactions betweenantigen and antibody include reversible noncovalent associations such aselectrostatic attraction, Van der Waals forces and hydrogen bonds.

Effector molecules, e.g., therapeutic, diagnostic, or detection moietiescan be linked to an antibody that specifically binds NGEP, using anynumber of means known to those of skill in the art. Exemplary effectormolecules include, but not limited to, a radiolabels, fluorescentmarkers, or toxins (e.g. Pseudomonas exotoxin (PE), see “MonoclonalAntibody-Toxin Conjugates: Aiming the Magic Bullet,” Thorpe et al.,“Monoclonal Antibodies in Clinical Medicine”, Academic Press, pp.168-190, 1982; Waldmann, Science, 252: 1657, 1991; U.S. Pat. Nos.4,545,985 and 4,894,443, for a discussion of toxins and conjugation).Both covalent and noncovalent attachment means may be used. Theprocedure for attaching an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain variety of functional groups; e.g., carboxylic acid(COOH), free amine (—NH₂) or sulfhydryl (—SH) groups, which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule. Alternatively, theantibody is derivatized to expose or attach additional reactivefunctional groups. The derivatization may involve attachment of any of anumber of linker molecules such as those available from Pierce ChemicalCompany, Rockford Ill. The linker can be any molecule used to join theantibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (e.g.,through a disulfide linkage to cysteine) or to the alpha carbon aminoand carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (e.g. enzymes or fluorescent molecules) drugs, toxins,and other agents to antibodies one skilled in the art will be able todetermine a suitable method for attaching a given agent to an antibodyor other polypeptide.

Therapeutic Methods and Pharmaceutical Compositions

An NGEP polypeptide can be administered to a subject in order togenerate an immune response. In one embodiment, a therapeuticallyeffective amount of an NGEP polypeptide is administered to a subject totreat prostate cancer. In exemplary applications, compositions areadministered to a patient suffering from a disease, such as prostatecancer, in an amount sufficient to raise an immune response toNGEP-expressing cells. Administration induces a sufficient immuneresponse to slow the proliferation of such cells or to inhibit theirgrowth. Amounts effective for this use will depend upon the severity ofthe disease, the general state of the patient's health, and therobustness of the patient's immune system. A therapeutically effectiveamount of the compound is that which provides either subjective reliefof a symptom(s) or an objectively identifiable improvement as noted bythe clinician or other qualified observer.

An NGEP polypeptide can be administered by any means known to one ofskill in the art (see Banga, A., Parenteral Controlled Delivery ofTherapeutic Peptides and Proteins, in Therapeutic Peptides and Proteins,Technomic Publishing Co., Inc., Lancaster, Pa., 1995) such as byintramuscular, subcutaneous, or intravenous injection, but even oral,nasal, or anal administration is contemplated. In one embodiment,administration is by subcutaneous or intramuscular injection. To extendthe time during which the peptide or protein is available to stimulate aresponse, the peptide or protein can be provided as an implant, an oilyinjection, or as a particulate system. The particulate system can be amicroparticle, a microcapsule, a microsphere, a nanocapsule, or similarparticle. (see, e.g., Banja, supra). A particulate carrier based on asynthetic polymer has been shown to act as an adjuvant to enhance theimmune response, in addition to providing a controlled release. Aluminumsalts may also be used as adjuvants to produce a humoral immuneresponse. Thus, in one embodiment, an NGEP polypeptide is administeredin a manner to induce a humoral response.

In another embodiment, an NGEP polypeptide is administered in a mannerto direct the immune response to a cellular response (that is, a CTLresponse), rather than a humoral (antibody) response. A number of meansfor inducing cellular responses, both in vitro and in vivo, are known.Lipids have been identified as agents capable of assisting in primingCTL in vivo against various antigens. For example, as described in U.S.Pat. No. 5,662,907, palmitic acid residues can be attached to the alphaand epsilon amino groups of a lysine residue and then linked (e.g., viaone or more linking residues, such as glycine, glycine-glycine, serine,serine-serine, or the like) to an immunogenic peptide. The lipidatedpeptide can then be injected directly in a micellar form, incorporatedin a liposome, or emulsified in an adjuvant. As another example, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine canbe used to prime tumor specific CTL when covalently attached to anappropriate peptide (see, Deres et al., Nature 342:561, 1989). Further,as the induction of neutralizing antibodies can also be primed with thesame molecule conjugated to a peptide which displays an appropriateepitope, the two compositions can be combined to elicit both humoral andcell-mediated responses where that is deemed desirable.

In yet another embodiment, to induce a CTL response to an immunogenicNGEP polypeptide or fragment thereof, a MHC class II-restricted T-helperepitope is added to the CTL antigenic peptide to induce T-helper cellsto secrete cytokines in the microenvironment to activate CTL precursorcells. The technique further involves adding short lipid molecules toretain the construct at the site of the injection for several days tolocalize the antigen at the site of the injection and enhance itsproximity to dendritic cells or other “professional” antigen presentingcells over a period of time (see Chesnut et al., “Design and Testing ofPeptide-Based Cytotoxic T-Cell-Mediated Immunotherapeutics to TreatInfectious Diseases and Cancer,” in Powell et al., eds., Vaccine Design,the Subunit and Adjuvant Approach, Plenum Press, New York, 1995).

A pharmaceutical composition including an NGEP polypeptide is thusprovided. In one embodiment, the NGEP polypeptide, or fragment thereof,is mixed with an adjuvant containing two or more of a stabilizingdetergent, a micelle-forming agent, and an oil. Suitable stabilizingdetergents, micelle-forming agents, and oils are detailed in U.S. Pat.Nos. 5,585,103; 5,709,860; 5,270,202; and 5,695,770, all of which areincorporated by reference. A stabilizing detergent is any detergent thatallows the components of the emulsion to remain as a stable emulsion.Such detergents include polysorbate, 80 (TWEEN)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,Zwittergent™ 3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, preferablyat about 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by BASF Wyandotte publications, e.g., Schmolka, J.Am. Oil. Chem. Soc. 54:110 (1977), and Hunter et al., J. Immunol.129:1244 (1981), PLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™1501, 150R1, 701, 901, 1301, and 130R1. The chemical structures of suchagents are well known in the art. In one embodiment, the agent is chosento have a hydrophile-lipophile balance (HLB) of between 0 and 2, asdefined by Hunter and Bennett, J. Immun. 133:3167 (1984). The agent canbe provided in an effective amount, for example between 0.5 and 10%,most preferably in an amount between 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the antigen in oil-in-water emulsion, i.e., to provide a vehicle forthe desired antigen, and preferably has a melting temperature of lessthan 65° C. such that emulsion is formed either at room temperature(about 20° C. to 25° C.), or once the temperature of the emulsion isbrought down to room temperature. Examples of such oils includesqualene, Squalane, EICOSANE™, tetratetracontane, glycerol, and peanutoil or other vegetable oils. In one specific, non-limiting example, theoil is provided in an amount between 1 and 10%, most preferably between2.5 and 5%. The oil should be both biodegradable and biocompatible sothat the body can break down the oil over time, and so that no adverseaffects, such as granulomas, are evident upon use of the oil.

An adjuvant can be included in the composition. In one embodiment, theadjuvant is a mixture of stabilizing detergents, micelle-forming agent,and oil available under the name Provax® (IDEC Pharmaceuticals, SanDiego, Calif.).

In another embodiment, a pharmaceutical composition includes a nucleicacid encoding an NGEP polypeptide or immunogenic fragment thereof. Atherapeutically effective amount of the NGEP polynucleotide can beadministered to a subject in order to generate an immune response. Inone specific, non-limiting example a therapeutically effective amount ofthe NGEP polynucleotide is administered to a subject to treat prostatecancer.

One approach to administration of nucleic acids is direct immunizationwith plasmid DNA, such as with a mammalian expression plasmid. Asdescribed above, the nucleotide sequence encoding NGEP, or animmunogenic peptide thereof, can be placed under the control of apromoter to increase expression of the molecule.

Immunization by nucleic acid constructs is well known in the art andtaught, for example, in U.S. Pat. No. 5,643,578 (which describes methodsof immunizing vertebrates by introducing DNA encoding a desired antigento elicit a cell-mediated or a humoral response) and U.S. Pat. Nos.5,593,972 and 5,817,637 (which describe operably linking a nucleic acidsequence encoding an antigen to regulatory sequences enablingexpression). U.S. Pat. No. 5,880,103 describes several methods ofdelivery of nucleic acids encoding immunogenic peptides or otherantigens to an organism. The methods include liposomal delivery of thenucleic acids (or of the synthetic peptides themselves), andimmune-stimulating constructs, or ISCOMS™, negatively charged cage-likestructures of 30-40 nm in size formed spontaneously on mixingcholesterol and Quil A™ (saponin). Protective immunity has beengenerated in a variety of experimental models of infection, includingtoxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ asthe delivery vehicle for antigens (Mowat and Donachie, Immunol. Today12:383, 1991). Doses of antigen as low as 1 μg encapsulated in ISCOMS™have been found to produce class I mediated CTL responses (Takahashi etal., Nature 344:873, 1990).

In another approach to using nucleic acids for immunization, an NGEPpolypeptide or an immunogenic peptide thereof can also be expressed byattenuated viral hosts or vectors or bacterial vectors. Recombinantvaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus,or other viral vectors can be used to express the peptide or protein,thereby eliciting a CTL response. For example, vaccinia vectors andmethods useful in immunization protocols are described in U.S. Pat. No.4,722,848. BCG (Bacillus Calmette Guerin) provides another vector forexpression of the peptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding an NGEP polypeptide or animmunogenic fragment thereof is introduced directly into cells. Forexample, the nucleic acid may be loaded onto gold microspheres bystandard methods and introduced into the skin by a device such asBio-Rad's Helios™ Gene Gun. The nucleic acids can be “naked,” consistingof plasmids under control of a strong promoter. Typically, the DNA isinjected into muscle, although it can also be injected directly intoother sites, including tissues in proximity to metastases. Doseages forinjection are usually around 0.5 μg/kg to about 50 mg/kg, and typicallyare about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Pat. No.5,589,466).

In addition, the cell growth inhibiting chimeric molecules including anantibody that specifically binds NGEP linked to a toxin (i.e., PE linkedto an anti-NGEP antibody), can be prepared in pharmaceuticalcompositions. These cell growth inhibiting molecules can be administeredby any method known to one of skill in the art. For example, to treatprostate cancer, the pharmaceutical compositions of this invention canbe administered directly into the prostate gland. Metastases of prostatecancer may be treated by intravenous administration or by localizeddelivery to the tissue surrounding the tumor.

The compositions for administration will commonly comprise a solution ofthe cell growth inhibiting chimeric molecules dissolved in apharmaceutically acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., buffered saline and thelike. These solutions are sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like. Theconcentration of cell growth inhibiting molecules in these formulationscan vary widely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs.

In one specific, non-limiting example, a pharmaceutical composition forintravenous administration, such as an immunotoxin, would be about 0.1to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg perpatient per day may be used, particularly if the agent is administeredto a secluded site and not into the circulatory or lymph system, such asinto a body cavity or into a lumen of an organ. Actual methods forpreparing administrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in such publicationsas Remingtons Phamaceutical Sciences, 19^(th) Ed., Mack PublishingCompany, Easton, Pa. (1995).

The compositions can be administered for therapeutic treatments. Intherapeutic applications, compositions are administered to a patientsuffering from a disease, such as prostate cancer, in a therapeuticallyeffective amount, which is an amount sufficient to cure or at leastpartially arrest the disease or a sign or symptom of the disease.Amounts effective for this use will depend upon the severity of thedisease and the general state of the patient's health. An effectiveamount of the compound is that which provides either subjective reliefof a symptom(s) or an objectively identifiable improvement as noted bythe clinician or other qualified observer.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In one embodiment, the dosage is administered once as a bolus,but in another embodiment can be applied periodically until either atherapeutic result is achieved. Generally, the dose is sufficient totreat or ameliorate symptoms or signs of disease without producingunacceptable toxicity to the patient.

Controlled release parenteral formulations of cell growth inhibitingchimeric molecules can be made as implants, oily injections, or asparticulate systems. For a broad overview of protein delivery systems(see Banga, A. J., Therapeutic Peptides and Proteins: Formulation,Processing, and Delivery Systems, Technomic Publishing Company, Inc.,Lancaster, Pa., 1995). Particulate systems include microspheres,microparticles, microcapsules, nanocapsules, nanospheres, andnanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres the therapeutic is dispersed throughoutthe particle. Particles, microspheres, and microcapsules smaller thanabout 1 μm are generally referred to as nanoparticles, nanospheres, andnanocapsules, respectively. Capillaries have a diameter of approximately5 μm so that only nanoparticles are administered intravenously.Microparticles are typically around 100 μm in diameter and areadministered subcutaneously or intramuscularly (see Kreuter, J.,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992); and Pec et al., J. Parent. Sci. Tech.44(2):58, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri, et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systemsfor controlled delivery of therapeutic proteins are known (e.g., U.S.Pat. Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,0284,957,735 and 5,019,369, 5,055,303; 5,514,670; 5,413,797; 5,268,164;5,004,697; 4,902,505; 5,506,206, 5,271,961; 5,254,342 and 5,534,496).

In another method, antigen presenting cells (APCs) are pulsed orco-incubated with peptides comprising an epitope from NGEP in vitro.These cells are used to sensitize CD8 cells, such as tumor infiltratinglymphocytes from prostate cancer tumors or peripheral blood lymphocytes.The TILs or PBLs preferably are from the subject. However, they shouldat least be MHC Class-I restricted to the HLA types the subjectpossesses. An effective amount of the sensitized cells are thenadministered to the subject.

PBMCs may be used as the responder cell source of CTL precursors. Theappropriate antigen-presenting cells are incubated with peptide, afterwhich the peptide-loaded antigen-presenting cells are then incubatedwith the responder cell population under optimized culture conditions.Positive CTL activation can be determined by assaying the culture forthe presence of CTLs that kill radio-labeled target cells, both specificpeptide-pulsed targets as well as target cells expressing endogenouslyprocessed forms of the antigen from which the peptide sequence wasderived.

The cells can be administered to inhibit the growth of cells of NGEPexpressing tumors. In these applications, a therapeutically effectiveamount of activated antigen presenting cells, or activated lymphocytes,are administered to a subject suffering from a disease, in an amountsufficient to raise an immune response to NGEP-expressing cells. Theresulting immune response is sufficient to slow the proliferation ofsuch cells or to inhibit their growth.

In a supplemental method, any of these immunotherapies is augmented byadministering a cytokine, such as IL-2, IL-3, IL-6, IL-10, IL-12, IL-15,GM-CSF, interferons.

Diagnostic Methods and Kits

A method is provided herein for the detection of NGEP-expressingprostate cells or prostate tissue in a biological sample. The sample canbe any sample that includes NGEP polypeptide or a nucleic acid encodingNGEP polypeptide. Such samples include, but are not limited to, tissuefrom biopsies, autopsies, and pathology specimens. Biological samplesalso include sections of tissues, such as frozen sections taken forhistological purposes. Biological samples further include body fluids,such as blood, sputum, serum, or urine. A biological sample is typicallyobtained from a mammal, such as a rat, mouse, cow, dog, guinea pig,rabbit, or primate. In one embodiment the primate is macaque,chimpanzee, or a human. In a further embodiment the subject has prostatecancer, or is suspected of having prostate cancer. Methods of detectioninclude, for example, radioimmunoassay, sandwich immunoassays (includingELISA), immunofluorescence assays, Western blot, affinity chromatography(affinity ligand bound to a solid phase), and in situ detection withlabeled antibodies.

In one embodiment, a method is provided for detecting an NGEPpolypeptide. Kits for detecting an NGEP polypeptide of fragment thereofwill typically comprise an antibody that specifically binds NGEP. Insome embodiments, an antibody fragment, such as an Fv fragment isincluded in the kit. For in vivo uses, the antibody is preferably anscFv fragment. In a further embodiment the antibody is labeled (e.g.fluorescent, radioactive, or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds an NGEP polypeptideor fragment thereof (e.g. for detection of NGEP expressing cells in asample). The instructional materials may be written, in an electronicform (e.g. computer diskette or compact disk) or may be visual (e.g.video files). The kits may also include additional components tofacilitate the particular application for which the kit is designed.Thus, for example, the kit may additionally contain means of detecting alabel (e.g. enzyme substrates for enzymatic labels, filter sets todetect fluorescent labels, appropriate secondary labels such as asecondary antibody, or the like). The kits may additionally includebuffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one embodiment of the present invention, the diagnostic kit comprisesan immunoassay. Although the details of the immunoassays may vary withthe particular format employed, the method of detecting an NGEPpolypeptide or fragment thereof in a biological sample generallycomprises the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toNGEP. The antibody is allowed to specifically bind under immunologicallyreactive conditions to form an immune complex, and the presence of theimmune complex (bound antibody) is detected directly or indirectly.

In an alternative set of embodiments, kits can be provided for detectingnucleic acids encoding NEGP or a fragment thereof in a biologicalsample. For example, sample from a subject can be tested to determinewhether nucleic acids encoding NGEP protein are present. In oneembodiment, an amplification procedure is utilized to detect nucleicacids encoding NGEP. In another embodiment, a blotting procedure (e.g.Northern Blot or Dot Blot) is used to detect the presence of nucleicacids encoding NGEP. Thus, a kit can include probes or primers thatspecifically hybridize to nucleic acids encoding NGEP.

In one embodiment, a kit provides a primer that amplifies nucleic acidencoding NGEP. Conveniently, the amplification is performed bypolymerase chain reaction (PCR). A number of other techniques are,however, known in the art and are contemplated for use. For example,Marshall, U.S. Pat. No. 5,686,272, discloses the amplification of RNAsequences using ligase chain reaction, or “LCR,” (Landegren et al.,Science 241:1077, 1988); Wu et al., Genomics, 4:569, 1989; Barany, inPCR Methods and Applications 1:5, 1991); and Barany, Proc. Natl. Acad.Sci. USA 88:189, 1991). Or, the RNA can be reverse transcribed into DNAand then amplified by LCR, PCR, or other methods. An exemplar protocolfor conducting reverse transcription of RNA is taught in U.S. Pat. No.5,705,365. Selection of appropriate primers and PCR protocols aretaught, for example, in Innis, M. et al., eds., PCR Protocols 1990(Academic Press, San Diego Calif.).

In one embodiment, the kit includes instructional materials disclosingmeans of use for the primer or probe. The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. The kits may additionally include buffers and otherreagents routinely used for the practice of a particular method. Suchkits and appropriate contents are well known to those of skill in theart.

The invention is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Computer Analysis of EST Sequences

The NCBI dbEST/CGAP database (see Emmert-Buck et al., Science 274:998,1996; Krizman, D. B. et al., Cancer Res. 56:5380, 1996); Strausberg, R.L. et al., Nat. Genet. 16:415, 1997), was used as a source for cDNAsequences. The ESTs from human tissues and tumors were downloaded fromthe NCBI EST database. The cDNA libraries that we processed are listedin several websites, including the Unigene Cluster from the NCBIwebsite, amongst others. The EST sequences were clustered and sorted asdescribed before (Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA95:300, 1998). However, the candidate gene list was updated by using theEST dataset of, May 2000. Two updated candidate lists were prepared, onewith the specificity cutoff for prostate of three as before and anotherwith the cutoff value of six.

The cluster CW-1 is identified by computer analysis of the human ESTdatabase as a prostate specific cluster. There are 5 ESTs in thiscluster of which 3 ESTs are from normal prostate and two ESTs are fromprostate cancer (FIG. 1). All ESTs are localized at chromosome 2q37.3 onhuman genome.

Example 2 Specificity of CW-1 Cluster

To determine the tissue specificity of the CW-1 cluster experimentally,a multi-tissue dot blot analysis was performed using a PCR generated DNAfragment from the cluster as a probe.

The human multiple tissue RNA blot (RNA Masterblot, Clontech, Palo Alto,Calif.) and Northern blot (Multiple Tissue Northern blot, Human II,Clontech) hybridizations were carried out as described previously (Liuet al., Biochem. Biophys. Res. Commun., 264:833, 1999). Briefly, RNAhybridizations with multiple tissue RNA dot-blot and northern blot wereperformed as follows: membranes were pre-hybridized for 2 h inhybridization solution (Hybrisol I; Intergen, Purchase, N.Y.) at 45° C.

The cDNA probe was produced from a PCR fragment generated using theprimer pair CW74: (SEQ ID NO: 3, AAG CTA GAC AGG CAG CAG GAC A) and CW75(SEQ ID NO:4, CATGAG AGA GCT GAG TGT GCA G; CW91: SEQ ID NO: 5, GCC TGATCT GCT CTG GGA CTC TGG), and was labeled with ³²P by random primerextension (Lofstrand Labs Ltd, Gaithersburg, Md.), added to themembranes and hybridized for 16 hours. The membranes were then washed2×15 minutes in 2×SSC, 0.1% SDS, at room temperature and then washed2×15 minutes in 0.5×SSC, 0.1% SDS, at 55° C. Finally the membranes wereexposed on x-ray film for 1-2 days. The washing and the autoradiographyof the blot were performed as described (Bera et al., Proc. Natl. Acad.Sci. USA 99: 3058-3063, 2002).

As shown in FIG. 2A, among the 76 different samples of normal and fetaltissue examined, CW-1 is detected strongly only in prostate sample. Theexpression of the CW-1 cluster is not detectable in any other tissuesincluding essential organs including brain (A1), heart (A4), lung (A8),kidney (A7), and pancreas (B9). Dot-blot analysis is not a verysensitive technique and often gave a negative signal for genes, whichare expressed at a very low level. To validate the result obtained fromthe dot-blot analysis, a more sensitive RT-PCR method was used for theexpression analysis of CW-1 cluster. A panel of cDNAs isolated fromseveral normal tissues including brain, heart, liver, lung, pancreas,colon, was used for PCR with a primer pair designed from the sequence ofthe CW-1 cluster. As shown in FIG. 2B, a specific band of 176 bp in sizeis detected only in prostate (two normal, lanes 3 and 12; two cancer,lanes 4 and 5) tissue indicating the CW-1 cluster is specificallyexpressed in prostate. Because of its specific expression in prostate,this sequence was named NGEP (New Gene Expressed in Prostate).

To determine the expression of NGEP in prostate cancer cell lines, anRT-PCR analysis was performed using RNAs from three prostate cancer cellline LnCAP, and PC3. NGEP expression was detected only in the RNA fromLnCAP cells but not in PC3 (FIG. 2D).

Example 3 Full Length cDNA Cloning of CW-1

To determine the transcript size of the CW-1 cluster in tissues, ananalysis was made of a Northern blot containing mRNAs from differenttissues including prostate. The PCR generated probe that was used forthe dot blot analysis was also used in this experiment. Methods aredescribed in Example 2.

As shown in FIG. 2C, several bands of different sizes are detected onlyin prostate lane. The smallest band, which likely is the mature mRNA, isabout 900 base pairs (bp) in size (arrow). The high molecular weightbands which are detected by the probe are probably unspliced variants ofthe transcript or transcripts with longer 3′ untranslated regions.

To isolate the full-length cDNA for CW-1 a 5′ and 3′ RACE PCR method wasemployed to isolate a clone of 917 bp in size (see FIG. 6).

The primers used in this study were synthesized by Lofstrand Lab,(Gaithersburg, Md.) and the nucleotide sequences of the primers are asfollows:

(SEQ ID NO: 6) Ngepex25, ACAGCA CCG TCC TGA TCG ATG TGA GC; (SEQ ID NO:7) Ngep3-2, TGT CTA GCT TCA GGT CCT CCT CCC AAA CG; (SEQ ID NO: 8) CW88,GCT TGC TGT CCA CCT GGC TCC GAG GC; (SEQ ID NO: 9) T420, AAA GAT AGA TCCTGC TCC AGG AGC CGG; (SEQ ID NO: 10) T421, GAC AGGTGA ATG GCA AAG GTGTCA GAG.

Rapid amplification of cDNA ends (RACE) was performed on Marathon Readynormal prostate cDNA (Clontech, Palo Alto, Calif.) and on total RNA froma prostate cancer sample (prepared using Trizol, Life Technologies) TheRACE PCR on the total RNA sample was made using the SMART RACE cDNAamplification kit (Clontech, Palo Alto, Calif.). The gene specificprimer CW88 was used for the RACE-PCR reaction with adopter primer AP1as recommended in the kit. The PCR product was gel purified and clonedinto the pCR2.1 TOPO TA cloning vector (Invitrogen, Carlsbad, Calif.).The positive clones were identified by restriction digestion using EcoRI and selective clones are sequenced using Perkin-Elmer's dRhodamineterminator sequencing kit (Perkin-Elmer Applied System, Warrington, UK).Finally, the full-length NGEP was cloned by PCR using the sequenceinformation from the RACE clones. The PCR primers used for thefull-length NGEP cloning were T420 and T421.

A 5′ and 3′ RACE PCR method was used to isolate a clone of 917 basepairs in size. The complete nucleotide sequence of the cDNA reveals thatit has an open reading frame of 179 amino acids (FIGS. 3 and 6). Thenucleotide sequence analysis of the full-length cDNA revels that it is anew gene with no significant sequence similarity with known genes in thedatabase. The calculated molecular weight of the protein encoded by theNGEP cDNA is about 19.6 kDa.

Example 4 NGEP mRNA is Expressed in Epithelial Cells of Normal Prostateand Prostate Cancer

To determine the cell types in normal prostate and prostate cancer thatexpress NGEP mRNA, in situ hybridization was performed using a biotinlabeled NGEP cDNA as a probe.

Briefly, biotinylated probes used for the in situ hybridization wereprepared using the full-length NGEP cDNA cloned in TOPO TA cloningvector. Full-length U6 cDNA cloned in same vector was used as positivecontrol. Probe labeling, pretreatment of the tissue section,hybridization, and washing conditions were similar to those describedpreviously (Bera et al., Proc. Natl. Acad. Sci. USA 99: 6997-7002,2002). Microscopic evaluation of the processed tissue sections wasdetermined by using a Nikon Eclipse 800 microscope.

As shown in FIG. 4, NGEP mRNA is highly expressed in prostaticepithelial cells of normal prostate and of prostate cancer specimens.There is no signal with a probe that does not contain the NGEP insert,indicating the specificity of the hybridization reaction. Also there wasno detectable signal in cells in the stromal compartment of the tissue,indicating that NGEP is specifically expressed in the epithelial cellsof the prostate. The signal intensity is comparable with the signalintensity of the positive control U6 probe. CD22, which is specificallyexpressed in B lymphocyte, was used as a negative control in theanalysis.

Example 5 In Vitro Transcription and Translation of the NGEP cDNA

The NGEP cDNA has a predicted open reading frame of 179 amino acids witha calculated molecular weight of 19.6 kDa. To determine the actual sizeof the protein encoded by the NGEP cDNA isolated, in vitro transcriptionand translation was performed using rabbit reticulocyte lysate system.

The complete NGEP transcript (SEQ ID NO:2), shown in FIG. 6, was clonedinto pBluescript II SK (+) (Stratagene) and sequenced. The PCR productwas directionally cloned into the pBluescript II SK (+) vector utilizingrestriction sites in the primers. This was examined in an in vitrotranscription coupled translation system using T7 RNA polymerase andrabbit reticulocyte lysate (TNT, Promega, Madison, Wis.). ³⁵S-Met (ICN,Costa Mesa, Calif.) was incorporated in the reaction for visualizationof translated products. The reaction was analyzed under reducingcondition on a polyacrylamide gel (16.5% Tris/Tricine, Bio-Rad) togetherwith a pre-stained marker (Life Technologies, Gaithersburg, Md.). Thegel was dried and subjected to fluororography.

SDS-PAGE analysis and fluorography of the translated product showed(FIG. 5A) that the NGEP cDNA encodes a protein product of about 20 kDain size (lane 2) and the size of the protein products from the in vitrotranscription and translation experiment agree with the predicted openreading frames of the cDNAs.

To determine the size of the protein encoded by NGEP in cells and itssubcellular localization in cells, LnCAP cells were transfected with aeukaryotic expression plasmid pcDNA3.1-Myc-His expressing NGEP with aMyc epitope tag at the carboxy terminus. LnCaP cells were maintained inRPMI medium 1640 (Quality Biologicals, Gaithersburg, Md.) at 37° C. with5% CO2. The medium is supplemented with 10% fetal bovine serum (QualityBiologicals), 2 mM L-glutamine, 1 mM sodium pyruvate, andpenicillin/streptomycin.

An eukaryotic expression plasmid pcDNA3.1-Myc-His expressing NGEP with aMyc epitope tag at the carboxy terminus was named pNGEP-Myc. pPATE-Mycwas also used as a positive control for Myc epitope (Bera et al., ibid).The LnCAP cells were transiently transfected with these plasmids. Aftertransfection, western blot analysis was done as described previously(Bera et al., ibid). Filters were probed with 200 ng/ml anti-Myc-tagmonoclonal (9E10; Santa Cruz Biotech, Santa Cruz, Calif.).

Western blot analysis of cell extracts transfected with plasmidpcDNA3.1-Myc-His expressing NGEP using anti-myc antibody detects a bandof 20 kDa (FIG. 5B, open arrow). In addition there is a high molecularweight band of about 24 kDa in size (FIG. 5B, closed arrow). Cellextracts transfected with pPATE-Myc-His (Bera et al., Proc. Natl. Acad.Sci. USA 99: 3058-3063, 2002) was used as positive control and theexpected 16 kDa band is detected (lane pPATE-myc). No band was detectedin untransfected cell extracts.

To determine the location of NGEP protein in the cell,immunocytochemistry was carried out in LnCAP cells transfected withplasmid pcDNA3.1-Myc-His expressing NGEP with a Myc epitope tag.

Co-transfection of pNGEP-Myc with pEGFP-C1 (Clontech) as a transfectionmarker was done in LnCAP cells. After 24 h incubation followingtransfection in a slide chamber, cells were fixed for 10 min in 4%formaldehyde, treated for 5 min with 0.2% triton X-100 in PBS forpermeability, blocked for 30 min with 0.4% normal goat globulin and 0.1%saponin in PBS, then incubated at room temperature for 1 h with 5 μg/mlanti-Myc-tag monoclonal. Subsequently, the cells were incubated at roomtemperature for 1 h with TRITC-conjugated secondary antibodies (JacksonLaboratories, Bar Harbor, Me.) then mounted in anti-fade solution withDAPI (Vector Laboratories, Burlingame, Calif.). Labeled cells wereanalyzed by laser confocal microscopy.

It was first confirmed that neither transfection with pEGFP alone orimmunostaining without anti-NGEP antibody showed positive signals ofanti-Myc antibody, whereas green signals of GFP as a transfection markerwere positive. As shown in FIG. 5C, immunostaining using anti-Mycantibody showed positive signals which are localized both in thecytoplasm and in the nucleus in LnCAP cells transfected with pNGEP-Myc(FIG. 5C). In about 5% of the transfected cells the red signals wasfound exclusively in the cytoplasm (FIG. 5D). The same results wereobtained in PC3 and 293T cell lines. These data indicate that NGEP islocalized in cytoplasm and nucleus and might be actively transportedbetween nucleus and cytoplasm or enter the nucleus passively.

Thus, a new gene, NGEP, has been identified. NGEP is specificallyexpressed in prostate cancer and normal prostate cells. NGEP islocalized at chromosome 2q37.3 on the human genome and has an openreading frame of 180 amino acids. The deduced amino acid sequence ofNGEP has no sequence similarity with any known proteins in the database.RT-PCR analysis of NGEP expression in three established prostate cancercell lines indicate that NGEP is expressed in androgen responsive LnCAPcells but not in either DU145 or PC3 cells which do not have androgendependency. Genetically, loss of 2q has been reported in clinicalsamples of prostate cancer (for example see Cher et al., Cancer Res. 56:3091-3102, 1996).

The NGEP cDNA has a predicted open reading frame of 179 amino acids witha calculated molecular weight of 19.6 kDa. Western blot analysis of NGEPtransfected cells detect an expected 20 kDa protein product. Inaddition, an additional band of 26 kDa in size was detected in celllysate transfected with NGEP (FIG. 4B, closed arrow). This 26 kDaproduct could be a phosphorylated form of NGEP. In fact, there areseveral predicted protein kinase phosphorylation sites in the NGEPsequence. The ExPAsy program predicts one cAMP-dependent protein kinasesite at position 78-81 (KRGS); one protein kinase C phosphorylation siteat position 138 (T×R); and three possible Casein kinase IIphosphorylation sites at positions 72 (SppE), 138 (TwrE) and 142 (TflD).Because of its distribution in both cytoplasm and in the nucleus, NGEPcould be a kinase itself.

Expression of NGEP is restricted to prostate cancer and normal prostatewith no detectable expression in any normal essential tissues we havetested In situ hybridization experiments using a prostate cancerprogression array showed that NGEP is expressed in more than 50% of thecancer specimens tested. Thus, NGEP can be used to detect prostatecancer, or as a target for immunotherapy.

Example 6 Radioimmunoassay to Detect NGEP

The following example sets forth an exemplary protocol for aradioimmunoassay to detect the presence NGEP in a sample.

Radiolabeling of NGEP

Two and a half micrograms of chemically synthesized or E. coli expressedNGEP are labeled with ¹²⁵I using the chloramine T method (Hunter andGreenwood, Nature 194:495, 1962). The labeled protein is then purifiedusing a PD-10 column (Amersham Pharmacia Biotech).

Anti-NGEP Antibody

Anti-NGEP antibodies are prepared by using proteinA purified antiserafrom rabbits immunized with a Pseudomonas exotoxin (PE)-NGEP fusionprotein using standard techniques (Bruggeman et al., BioTechniques10:202, 1992).

Standard Curve

A standard curve is established by mixing a fixed amount of labeled NGEP(˜0.2 ng at about 170 μCi/μg) with different concentrations of unlabeledNGEP (0.1 ng-50 ng) in 250 μl buffer (PBS with 0.25% bovine serumalbumin) containing 1 μg of anti-NGEP antibody. The samples areincubated at room temperature for 4 hours. ProteinA sepharose beads areadded and incubated for another hour. Finally the beads are collected bycentrifugation and washed with buffer 3 times. The remaining bead pelletis measured for radioactivity in a gamma counter.

Sample Measurement

To measure the amount of NGEP in a tissue extract or a protein extractfrom a cell culture the same procedure is used, but with the samplesubstituted for the known amounts of the protein used in the standardcurve description.

Example 7 Production of an Immune Response Against NGEP in a Primate

The prostate gland of the rhesus monkey is structurally and functionallysimilar to the human prostate (Wakui et al., J. Anat. 181:121, 1992;U.S. Pat. No. 6,165,460). Thus, juvenile male rhesus monkeys (Macacamulatta), ages 1 to 2 years, are assigned to (e.g., three vaccinationgroups of four animals each, a low dose, a high dose and a controlgroup). One animal from each group is surgically prostatectomized toparallel two situations with regard to potential therapy in humans: (a)prostate intact, with primary and/or metastatic disease; or (b) patientsprostatectomized with prostate cancer metastatic deposits. Animals areimmunized 3 times over a two month period with a recombinant virus (e.g.a pox virus, see U.S. Pat. No. 6,165,460). For example, a dose of either1×10⁷ or 1×10⁸ PFU of a recombinant pox virus encoding NGEP isadministered to 4 animals by skin scarification. A control vector (e.g.V-Wyeth, 1×10⁸ PFU) is administered to a control group of animals.

Physical examinations are performed on ketamine (Ketamine® HCl, 10 mg/kgI.M.) sedated animals. Rectal temperatures and weights are recorded foreach monkey on a weekly basis. The vaccination site is observed anderythema and swelling of the vaccination site are measured by caliper.Each animal is examined for regional lymphadenopathy, hepatomegaly, andsplenomegaly. Any other gross abnormalities were also recorded.

Blood is obtained by venipuncture from the femoral vein of ketaminesedated animals before and after each immunization. A complete bloodcount, differential, hepatic and renal chemistry evaluation is performedon each monkey. Results are compared to normal primate values.Circulating levels of NGEP before and after immunization are analyzed(e.g by immunoassay or Northern blot).

Prior to each immunization and 2 weeks following each immunization,anti-NGEP antibody is quantified by ELISA. Microtiter plates are coatedwith purified NGEP (e.g. 100 ng/well), ovalbumin (100 ng/well, Sigma),or 1×10⁷ PFU/well UV-inactivated V-Wyeth in phosphate buffered saline(PBS). The plates are blocked (e.g. using 2% BSA in PBS), dried, andstored at −20° C. until used. The plates are incubated with serum (e.gdiluted 1:5), as well as a monoclonal antibody for NGEP as a standardcontrol, for 24 hours at 4° C. Plates are washed several times (e.g.with PBS containing 1% BSA), and incubated with a commercially labeledantibody that specifically binds the anti-NGEP monoclonal antibody. Anappropriate reagent system is used to visualize antibody binding. Forexample the antibody is labeled with horseradish peroxidase (HRP), anddetected by HRP substrate system (Kirkegaard & Perry Laboratories,Gaithersburg, Md.) according to the manufacture's instructions. Theabsorbance of each well is read at 405 nm using a Bio-Tek EL310microplate ELISA reader (Winooski, Vt.).

Sera from each monkey is analyzed by ELISA for immunoreactivity to NGEP.Sera obtained from monkeys prior to vaccination are also analyzed, andare negative for reactivity to NGEP. NGEP specific T-cell responses inmonkeys immunized with NGEP containing vector or control vector are alsoanalyzed using a lymphoproliferative assays using peripheral bloodmononuclear cells.

Example 8 Kit for the Detection of Metastatic Prostate Cancer

Prostate cancer is known to metastasize to other areas of the body, suchas bone. Antibodies to an NGEP polypeptide can be used to detectprostate cancer cells at locations other than the prostate.

In order to determine if a metastatic tumor originates in the prostate,the expression of NGEP is assessed. Specifically, a kit is utilized thatprovides an immunoassay that can be used to confirm that the cancercells are of prostate origin.

A biological sample of the metastasis is obtained. In one example, thesample is a bone marrow sample. Non-specific immunoreactive sites onbiological sample are blocked with a commercially available blockingagent, such as 10% bovine serum albumin in phosphate buffered saline(PBS), for thirty minutes at room temperature. The sample is thencontacted with a mouse monoclonal antibody that specifically binds NGEPfor an incubation period sufficient to allow formation of an immunecomplex (e.g. ten minutes to three hours at room temperature in asolution of 1% BSA). The presence of the immune complex (bound antibody)is detected by incubating the sample with a commercially availablelabeled secondary antibody that specifically binds the NGEP antibody.For example, a fluorescent labeled (e.g. fluorescein isothiocyanate,FITC) goat anti-mouse antibody is diluted 1:100 in PBS/1% BSA andincubated with the sample for an amount of time sufficient to form animmune complex (e.g. ten minutes to about two hours at roomtemperature). The samples are then processed to determine binding of thesecond antibody (e.g. detection of fluorescence). A positive signalindicates that the metastasis is of prostate origin.

Example 9 Activation of T Cells Using NGEP

Methods for evaluating immunogenicity of peptides are known.Immunogenicity be evaluated by, for example, evaluation of primary Tcell cultures from normal individuals (see, e.g., Wentworth et al., Mol.Immunol. 32:603, 1995; Celis, et al., Proc. Natl. Acad. Sci. USA91:2105, 1994; Tsai et al., J. Immunol. 158:1796 (1997); Kawashima etal., Human Immunol. 59:1 (1998)); by immunization of HLA transgenic mice(see, e.g., Wentworth et al., J. Immunol. 26:97, 1996; Wentworth et al.,Int. Immunol. 8:651, 1996; Alexander, et al., J. Immunol. 159:4753,1997), and by demonstration of recall T cell responses from patients whohave been effectively vaccinated or who have a tumor; (see, e.g.,Rehermann et al., J. Exp. Med. 181:1047, 1995; Doolan et al., Immunity7:97, 1997; Bertoni et al., J. Clin. Invest. 100:503 (1997); Threlkeld,S. C. et al., J. Immunol. 159:1648 1997; Diepolder et al., J. Virol.71:6011 1997).

In choosing CTL-inducing peptides of interest, peptides with higherbinding affinity for class I HLA molecules can be utilized. Peptidebinding is assessed by testing the ability of a candidate peptide tobind to a purified HLA molecule in vitro.

Based on the polypeptide sequence of NGEP, amino acid sequences bearingmotifs for any particular HLA molecule can be identified. Peptidesincluding these motifs can be prepared by any of the typical methods(e.g., recombinantly, chemically, etc.). Because NGEP is a self protein,the amino acid sequences bearing HLA binding motifs are those thatencode subdominant or cryptic epitopes. Those epitopes are identified bya lower comparative binding affinity for the HLA molecule with respectto other epitopes in the molecule or compared with other molecules thatbind to the HLA molecule.

Polypeptides that include an amino acid sequence from NGEP that, inturn, include an HLA binding motif also are useful for eliciting animmune response. This is because, in part, such proteins will beprocessed by the cell into a peptide that can bind to the HLA moleculeand that have an NGEP epitope.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus et al., Cell 47:1071, 1986;Babbitt et al., Nature 317:359, 1985; Townsend and Bodmer, Annu. Rev.Immunol. 7:601, 1989; Germain, Annu. Rev. Immunol. 11:403, 1993).Through the study of single amino acid substituted antigen analogs andthe sequencing of endogenously bound, naturally processed peptides,critical residues that correspond to motifs required for specificbinding to HLA antigen molecules have been identified (see, e.g.,Southwood et al., J. Immunol. 160:3363, 1998; Rammensee et al.,Immunogenetics 41:178, 1995; Rammensee et al., J. Curr. Opin. Immunol.10:478, 1998; Engelhard, Curr. Opin. Immunol. 6:13 (1994); Sette andGrey, Curr. Opin. Immunol. 4:79, 1992).

Furthermore, x-ray crystallographic analysis of HLA-peptide complexeshas revealed pockets within the peptide binding cleft of HLA moleculeswhich accommodate, in an allele-specific mode, residues borne by peptideligands; these residues in turn determine the HLA binding capacity ofthe peptides in which they are present (e.g., Madden, Annu. Rev.Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont etal., Immunity 8:305, 1998; Stem et al., Structure 2:245, 1994; Jones,Curr. Opin. Immunol. 9:75, 1997; Brown, et al., Nature 364:33, 1993).

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within NGEP that have the potential of binding particular HLAmolecules.

One method of identifying genes encoding antigenic determinants is asfollows: Tumor infiltrating lymphocytes (TILs) from a subject withprostate cancer are grown and tested for the ability to recognize theautologous cancer in vitro. These TILs are administered to the subjectto identify the ones that result in tumor regression. The TILs are usedto screen expression libraries for genes that express epitopesrecognized by the TILs. Subjects then are immunized with these genes.Alternatively, lymphocytes are sensitized in vitro against antigensencoded by these genes. Then the sensitized lymphocytes are adoptivelytransferred into subjects and tested for their ability to cause tumorregression. Rosenberg, et al., Immunol. Today 1997 18:175 (1997).

To ensure that NGEP elicits a CTL response to NGEP in vivo (or, in thecase of class II epitopes, elicits helper T cells that cross-react withthe wild type peptides), the NGEP can be used to immunize T cells invitro from individuals of the appropriate HLA allele. Thereafter, theimmunized cells' capacity to induce lysis of NGEP-sensitized targetcells is evaluated.

More generally, NGEP peptides can be synthesized and tested for theirability to bind to HLA proteins and to activate HTL or CTL responses, orboth.

Conventional assays utilized to detect T cell responses includeproliferation assays, lymphokine secretion assays, direct cytotoxicityassays, and limiting dilution assays. For example, antigen-presentingcells that have been incubated with a peptide can be assayed for theability to induce CTL responses in responder cell populations.

PBMCs can be used as the responder cell source of CTL precursors. Theappropriate antigen-presenting cells are incubated with peptide, afterwhich the peptide-loaded antigen-presenting cells are then incubatedwith the responder cell population under optimized culture conditions.Positive CTL activation can be determined by assaying the culture forthe presence of CTLs that kill radio-labeled target cells, both specificpeptide-pulsed targets as well as target cells expressing endogenouslyprocessed forms of the antigen from which the peptide sequence wasderived.

A method which allows direct quantification of antigen-specific T cellsis staining with Fluorescein-labeled HLA tetrameric complexes (Altman etal., Proc. Natl. Acad. Sci. USA 90:10330, 1993); Altman et al., Science274:94, 1996). Alternatively, staining for intracellular lymphokines,interferon-γ release assays or ELISPOT assays, can be used to evaluateT-cell responses.

CTL activation may be assessed using such techniques known to those inthe art such as T cell proliferation and secretion of lymphokines, e.g.IL-2 (see, e.g. Alexander et al., Immunity 1:751-761 (1994)).

In one specific, non-limiting example, transgenic mice that express achimeric human class 1 major histocompatibility (MHC) class 1 moleculecomposed of the α1 and α2 domains of HLA-A2.1 and the α3, transmembraneand cytoplasmic domains of H2-kb HLA transgenic mice (see, e.g.,Wentworth, P. A. et al., J. Immunol. 26:97, (1996); Wentworth, P. A. etal., Int. Immunol. 8:651 (1996); Alexander, J. et al., J. Immunol.159:4753 (1997)) are immunized with plasmid DNA encoding NGEP.Specifically, each mouse is injected intramuscularly with 100 μg ofplasmid five times every three weeks. After the final immunization CD8+cells are partially purified using antibody-coated magnetic beads andre-stimulated with syngeneic spleenocytes for one week in T-stim media.Specifically cells from the immunized transgenic mice are co-culturedeither with stimulating spleenocytes (e.g., 3.5×10⁶ spleenocytes) pulsedwith various concentrations (100, 0.1 or 0.0001 μM) NGEP peptide or inthe presence of free peptide (1 μM) in a 24 well plate containing 2 mlof a 1:1 mixture of RPMI1640 mdeia and Eagle-Hanks amino acid mediumsupplemented with L-glutamine, sodium pyruvate, non-essential aminoacids, antibiotics (penicillin, streptomycin, 5×10⁻⁵ M 2-mercaptoetanol,10% fetal calf serum, and 10% T-stim (Collaborative Biomedical Products,Bedford, Mass.). On day seven, a cytoxoic T lymphocyte (CTL) assay iscarried out using LnCAP cells expressing NGEP as target cells in thepresence of peptides fragments of NGEP. For example, LnCAP target cells(1×10⁶) are labeled with 300 μCi of Na₂ ⁵¹CrO₄ in 200-250 μl for 2 hoursat 37° C. For test samples, targets are pulsed with peptide duringlabeling. Cells are then washed and added to wells along with theappropriate number of effector cells in 96-well round bottom plates.After four hours, supernatents are harvested and counted in an ISOMEDICgamma counter (ICN). The mean of triplicate samples and percent of ⁵¹Crrelease is calculated (see Alexander-Miller et al, Proc. Natl. Acad.Sci. USA 93: 4102). The results demonstrate that NGEP peptides can beused to induce cytotoxic activity against LnCAP cells.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. An isolated Novel Gene Expressed in Prostate (NGEP) polypeptidehaving an amino acid sequence selected from the group consisting of: (1)an amino acid sequence 80% homologous to SEQ ID NO:1; (2) a conservativevariant of SEQ ID NO:1; (3) an immunogenic fragment comprising eightconsecutive amino acids of SEQ ID NO:1 that specifically binds to anantibody that specifically binds SEQ ID NO:1; and (4) SEQ ID NO:1.
 2. Anisolated nucleic acid sequence encoding the polypeptide of claim
 1. 3.The isolated nucleic acid sequence of claim 2, comprising a sequence asset forth as SEQ ID NO:2, or a degenerate variant thereof.
 4. Theisolated nucleic acid sequence of claim 3, operably linked to apromoter.
 5. An expression vector comprising the nucleic acid sequenceof claim
 2. 6. An isolated host cell transfected with the nucleic acidsequence of claim
 2. 7. The host cell of claim 6, wherein the host cellis a mammalian cell.
 8. An isolated antibody that specifically binds thepolypeptide of claim
 1. 9. The antibody of claim 8, wherein the antibodyis a monoclonal antibody.
 10. The antibody of claim 8, comprising adetectable label.
 11. The antibody of claim 8, conjugated to a toxin.12. A method for detecting prostate cancer in a subject, comprisingcontacting a sample obtained from the subject with the antibody of claim8 for a sufficient amount of time to form an immune complex; detectingthe presence the immune complex, wherein the presence of an immunecomplex demonstrates the presence of prostate cancer in the subject. 13.The method of claim 12, wherein the sample is a biopsy, blood, serum, orurine sample.
 14. The method of claim 12, wherein the sample is a biopsysample of non-prostate origin.
 15. A method for detecting a prostatecancer in a subject, comprising detecting the expression of the NGEPpolypeptide of claim 1 in a sample from the subject, wherein an increasein the expression of the NGEP as compared to a control indicates thepresence of the prostate cancer.
 16. The method of claim 15, whereindetecting the expression of NGEP polypeptide comprises contacting thesample with an antibody that specifically binds the NGEP polypeptide fora sufficient amount of time to form an immune complex; and detecting thepresence of the immune complex.
 17. The method of claim 15, whereindetecting the expression of NGEP polypeptide comprises detecting thepresence of mRNA encoding NGEP polypeptide.
 18. The method of claim 15,wherein detecting the presence of mRNA encoding NGEP polypeptidecomprises a Northern Blot analysis, an RNA dot blot, or a reversetranscriptase polypermase chain reaction (RT-PCR) assay.
 19. A methodfor producing an immune response against a cell expressing NGEP in asubject, comprising administering to the subject a therapeuticallyeffective amount of the NGEP polypeptide of claim 1, or a polynucleotideencoding the NGEP polypeptide, thereby producing the immune response.20. The method of claim 19, wherein the subject has prostate cancer. 21.The method of claim 19, wherein the immune response decreases the growthof the prostate cancer.
 22. A method for inhibiting the growth of amalignant cell expressing NGEP, comprising, (i) culturing cytotoxic Tlymphocytes (CTLs) or CTL precursor cells with the polypeptide of claim1 to produce activated CTLs or CTL precursors that recognize an NGEPexpressing cell, and (ii) contacting the malignant cell with theactivated CTLs or CTLs matured from the CTL precursors, therebyinhibiting the growth of the malignant cell.
 23. A method for inhibitingthe growth of a malignant cell expressing NGEP, comprising: contactingthe malignant cell with an effective amount of a cell-growth inhibitingmolecule, wherein the cell growth inhibiting molecule comprises theantibody of claim 8 and an effector molecule, thereby inhibiting thegrowth of the malignant cell.
 24. The method of claim 23, wherein theeffector molecule is a chemotherapeutic agent.
 25. The method of claim23, wherein the effector molecule comprises a toxic moiety.
 26. Themethod of claim 23, wherein the toxic moiety is selected from the groupconsisting of ricin A, abrin, diphtheria toxin or a subunit thereof,Pseudomonas exotoxin or a portion thereof, and botulinum toxins Athrough F.
 27. The method of claim 26, wherein the Pseudomonas exotoxinis selected from the group consisting of PE35, PE37, PE38, and PE40. 28.A pharmaceutical composition comprising a therapeutically effectiveamount of the polypeptide of claim 1, a polynucleotide encoding thepolypeptide, or an antibody that specifically binds the polypeptide, ina pharmaceutically acceptable carrier.
 29. A method for reducing thenumber of prostate cancer cells in a subject, comprising administeringto the subject a therapeutically effective amount of the NGEPpolypeptide of claim 1, a therapeutically effective amount of apolynucleotide encoding the NGEP polypeptide, or an antibody thatspecifically binds the polypeptide, wherein the administration of theNGEP polypeptide, the polynucleotide or the antibody, results in animmune response to NGEP, thereby reducing the number of prostate cancercells in the subject.
 30. A method for reducing the number of prostatecancer cells in a subject, comprising administering to the subject atherapeutically effective amount of the antibody of claim 8, therebyreducing the number of prostate cancer cells in the subject.
 31. A kitfor a detection assay, comprising (1) a container comprising an isolatednucleic acid sequence of at least ten nucleotides in length thatspecifically binds to SEQ ID NO:2 under highly stringent hybridizationconditions; (2) a container comprising the isolated antibody of claim 8;or (3) a container comprising an isolated nucleic acid sequence of atleast ten nucleotides in length that specifically binds to SEQ ID NO:2under highly stringent hybridization conditions and a containercomprising the isolated antibody of claim 8; and further comprising (4)instructions for the use of the isolated nucleic acid sequence or theantibody.